Cryo-EM structures of type IV pili complexed with nanobodies reveal immune escape mechanisms

Type IV pili (T4P) are prevalent, polymeric surface structures in pathogenic bacteria, making them ideal targets for effective vaccines. However, bacteria have evolved efficient strategies to evade type IV pili-directed antibody responses. Neisseria meningitidis are prototypical type IV pili-expressing Gram-negative bacteria responsible for life threatening sepsis and meningitis. This species has evolved several genetic strategies to modify the surface of its type IV pili, changing pilin subunit amino acid sequence, nature of glycosylation and phosphoforms, but how these modifications affect antibody binding at the structural level is still unknown. Here, to explore this question, we determine cryo-electron microscopy (cryo-EM) structures of pili of different sequence types with sufficiently high resolution to visualize posttranslational modifications. We then generate nanobodies directed against type IV pili which alter pilus function in vitro and in vivo. Cyro-EM in combination with molecular dynamics simulation of the nanobody-pilus complexes reveals how the different types of pili surface modifications alter nanobody binding. Our findings shed light on the impressive complementarity between the different strategies used by bacteria to avoid antibody binding. Importantly, we also show that structural information can be used to make informed modifications in nanobodies as countermeasures to these immune evasion mechanisms.

This is an interesfing combinafion of cryoEM, MD and funcfional studies focused on the mechanisms of Neisseria meningidifis to introduce variafions in the surface of its pilus, which helps to explain why thus far the search for an anfibody against the pilus had liftle success.The authors present first three cryo-EM structures of Type IV pilus (T4P) class I corresponding to three subvariants.In addifion, they generated two nanobodies against T4P and obtained three addifional high-resolufion cryo-EM structures of two of the subvariants with a nanobody bound.The cryo-EM structures appear to have good resolufion which resolved sequence variafions and posftranslafional modificafions, supporfing the conclusions.Novel ideas are that the combinafion of sequence variafions and post translafional modificafions alters the pilus surface, which affects anfibody recognifion, and that a combinafion of camelid nanobodies could be a successful strategy to combat immune evasion.Overall, the reviewer thinks that the manuscript is worthy of publicafion in Nature Communicafions but there remain concerns on the quality of the figures as well as the precision of the wrifing as indicated below that should be addressed.
1.The manuscript is not wriften for the general public as the authors rely heavily on the jargon in the field.Please edit to aftract the interest of the more general reader.
2. The introducfion is not well focused and lacks clarity.It should state that only class I will be examined, which variants, and why.
3. Methods: the number of experimental replica are not indicated in most secfions, this informafion needs to be included.

Results:
The six reconstrucfions reach good resolufion and support the results, but several pieces of informafion are missing:   7.Many sentences in the discussion are too vague which reduces the interest and does not convey much informafion.Examples of such sentences are "could impact anfibody binding", "significantly impacts anfibody recognifion "(but how?), has "strong implicafion", "substanfial influence", etc. Please subsfitute with informafive sentences.

OTHER SPECIFIC COMMENTS:
Line 230: "In the case of F10, CDR1 does not seem to contribute significantly to the F10 binding, it remains on the side of the nanobody."Please define CDR and as it is part of the nanobody, it is not surprising that remains on its side; probably the authors mean something else.
Line 319: "Interesfingly strand b1 of the beta sheet strand parallel to the alpha-beta loop is also submifted to several amino acid changes the importance of which was previously not recognized."Improve the sentence and add the residue range for clarity.
Line 340-342: "The structure of the F10-pilus complex shows the high specificity of the VHH for the SB hypervariable loop, but in exchange this VHH avoids variability in the region close to G3P." Please improve sentence.
Page 45 does not seem to belong to the manuscript.
Reviewer #2 (Remarks to the Author): In the manuscript "Cryo-EM structures of type IV pili complexed with nanobodies reveal immune escape mechanisms" the authors provide a detailed structure-based analysis of Neisseria meningifidis immune escape mechanisms by obtaining cryoEM structures of type IV pilus variants in the absence and presence of bound nanobodies.The cryoEM reconstrucfions are high quality, at resolufions suitable to idenfify posftranslafional modificafions and amino acid differences that contribute to immune escape.They show how anfibodies bind to variable surfaces like the PTMs and hypervariable loop of the major pilin, and how small changes in these can disrupt binding.They further show that these anfi-pilin nanobodies, when fused with mouse Fcs, can prevent N. meningifidis growth in the mouse model, demonstrafing how an understanding of these molecular processes can be of potenfial therapeufic value.Given the hypervariability in the major pilin the authors might comment in the Discussion on how the type IV pilus is such an effecfive adhesion.Most of my addifional comments are editorial in nature.
Line 61-63 -It should be noted that not all type IV pili exhibit surface variafion.
Line 66072 -Say something about the prevalence of Class I vs II strains (as in Line 355).
Line 110 -"isolated segments extracted and curated" Please explain "curated" and indicate that the filaments that show well resolved structural features are class averages, not individual pilus filaments.Similarly, in the legend for the figure in quesfion, (line 770)-"2D classes" should be "2D class averages".In this same line remove "high resolufion" as pilin monomers and secondary structure are not high (i.e.atomic) resolufion features.
Line -OG, OE1 -use Greek lefters for G and E here and elsewhere.
Line -"cycle" should be "ring" Line 142-153 -Clarify that the only differences between SA and SB pili are in sequence, not PTMs.
Line -Indicate from which strain the pili were purified -8013 SB? Cite Fig. S1c instead of S1b.
Line -Change "the same procedure was conducted" to "binding was also tested" ("procedure" sounds like the phage selecfion procedure).
Line 197-197 -Change "the effect of nanobodies on a possible neutralizafion of" to "the ability of nanobodies to neutralize".
Line -Report nanobody concentrafion using the same units here and in the figure .Line -For clarity include "phagocyfic", i.e. "phagocyfic Kupffer cells" (and correct the spelling of Kupffer here and in Line 355).
Line -Delete "which is not a variable area".Line -Change "cycle" to "ring".
Line -"pics" should be "peaks" Line -Is the alpaca's name relevant?(and Alpaca need not be capitalized).
Line -Correct spelling of peroxidase.
Line -Variable amino acids are shown in green -is this regardless of whether or not the changes are conserved?Recommend coloring only non-conserved changes.Line -Correct "phospohoglycerol".Line 844 -"indicate" Fig. S4 -Some of the text is small and faint so hard to read.Supplementary tables are not labeled.Something is wrong with the formafting.

Reviewer #3 (Remarks to the Author):
This work produced the cryo-electron microscopy structures of pili of different sequence types with sufficiently high resolufion to visualize posftranslafional modificafions and their complexes with nanobodies.Molecular dynamics simulafions contributed to the structure modeling and refinement.
While liftle simulafion data was presented to validate the findings, the supplementary videos provide some details.The simulafion protocol seems alright.The video S1 shows the membrane with a 16-mer pilus inserted keeps moving up, or the 16-mer keeps inserfing deeper, unfil the final quarter of the simulafion when G3P touches the membrane.This does not invalidate the simulafion, but does show that the inifial inserfion of the pilus is not right.

Point by point response for Nature Communications manuscript NCOMMS-23-47033-T REVIEWER COMMENTS
Reviewer #1 (Remarks to the Author): This is an interesting combination of cryoEM, MD and functional studies focused on the mechanisms of Neisseria meningiditis to introduce variations in the surface of its pilus, which helps to explain why thus far the search for an antibody against the pilus had little success.The authors present first three cryo-EM structures of Type IV pilus (T4P) class I corresponding to three subvariants.In addition, they generated two nanobodies against T4P and obtained three additional high-resolution cryo-EM structures of two of the subvariants with a nanobody bound.The cryo-EM structures appear to have good resolution which resolved sequence variations and posttranslational modifications, supporting the conclusions.Novel ideas are that the combination of sequence variations and post translational modifications alters the pilus surface, which affects antibody recognition, and that a combination of camelid nanobodies could be a successful strategy to combat immune evasion.Overall, the reviewer thinks that the manuscript is worthy of publication in Nature Communications but there remain concerns on the quality of the figures as well as the precision of the writing as indicated below that should be addressed.
1.The manuscript is not written for the general public as the authors rely heavily on the jargon in the field.Please edit to attract the interest of the more general reader.
Efforts have been made to reduce topic-specific language and make the text accessible to scientist from other areas.In particular, the beginning of the introduction has been rewritten to fit a broader public.
b) # of filaments analyzed and # of filaments discarded for each dataset.

The particles used and discarded can be consulted in Supplementary Table 1, along with collection details and refinement statistics. The table has been modified for better clarity.
c) Please specify whether the 2D classification produce different classes, or was 3D used to select the best classes?2D classification was extensively used to clean the particles and yielded wellcentered, vertically aligned homogeneous filaments or diverse orientations.In all cases, heterogeneous refinement or 3D classification attempts did not yield alternative conformations or large quantities of low-quality particles, therefore virtually all particles selected during the 2D classification step were used for helical refinement.The text has been modified accordingly.7.Many sentences in the discussion are too vague which reduces the interest and does not convey much information.Examples of such sentences are "could impact antibody binding", "significantly impacts antibody recognition "(but how?), has "strong implication", "substantial influence", etc. Please substitute with informative sentences.Several modifications have been made in the discussion to address this point.

OTHER SPECIFIC COMMENTS:
Line 230: "In the case of F10, CDR1 does not seem to contribute significantly to the F10 binding, it remains on the side of the nanobody."Please define CDR and as it is part of the nanobody, it is not surprising that remains on its side; probably the authors mean something else.The acronym CDR was spelled out and sentence clarified.
Line 319: "Interestingly strand b1 of the beta sheet strand parallel to the alpha-beta loop is also submitted to several amino acid changes the importance of which was previously not recognized."Improve the sentence and add the residue range for clarity.

This point was clarified
Line 340-342: "The structure of the F10-pilus complex shows the high specificity of the VHH for the SB hypervariable loop, but in exchange this VHH avoids variability in the region close to G3P." Please improve sentence.

The sentence was rephrased to clarify
Lines 662, 664: please describe the "hysteresis threshold" and "sinuosity threshold".

A definition has been included in the text
Page 45 does not seem to belong to the manuscript.I am not sure which page this corresponds to, there does not seem to be any extra page in the PDF that was included on our side, perhaps this results from the submission system?
Reviewer #2 (Remarks to the Author): In the manuscript "Cryo-EM structures of type IV pili complexed with nanobodies reveal immune escape mechanisms" the authors provide a detailed structure-based analysis of Neisseria meningitidis immune escape mechanisms by obtaining cryoEM structures of type IV pilus variants in the absence and presence of bound nanobodies.The cryoEM reconstructions are high quality, at resolutions suitable to identify posttranslational modifications and amino acid differences that contribute to immune escape.They show how antibodies bind to variable surfaces like the PTMs and hypervariable loop of the major pilin, and how small changes in these can disrupt binding.They further show that these anti-pilin nanobodies, when fused with mouse Fcs, can prevent N. meningitidis growth in the mouse model, demonstrating how an understanding of these molecular processes can be of potential therapeutic value.Given the hypervariability in the major pilin the authors might comment in the Discussion on how the type IV pilus is such an effective adhesion.Most of my additional comments are editorial in nature.
Line 61-63 -It should be noted that not all type IV pili exhibit surface variation.In pathogenic Neisseria species all strains express pili with potential surface variation, due to amino acid changes and/or PTMs such as sugars.Type IV pili from other bacterial species can indeed exhibit no or moderate surface variation.The text was modified to clarify that statements referred to Neisseria spp.
Line 66072 -Say something about the prevalence of Class I vs II strains (as in Line 355).A sentence has been added.
Line 99 -I recommend using "nanobodies" instead of the cryptic "VHH" acronym.The term "VHH" was replaced by "nanobody" throughout the manuscript Line 110 -"isolated segments extracted and curated" Please explain "curated" and indicate that the filaments that show well resolved structural features are class averages, not individual pilus filaments.Similarly, in the legend for the figure in question, (line 770)-"2D classes" should be "2D class averages".In this same line remove "high resolution" as pilin monomers and secondary structure are not high (i.e.atomic) resolution features.

The sentence has been reworded and the suggested corrections in the legend made
Line 131 -OG, OE1 -use Greek letters for G and E here and elsewhere.

Greek letter have been introduced
Line 134 -"cycle" should be "ring" Replaced Line 142-153 -Clarify that the only differences between SA and SB pili are in sequence, not PTMs.

A sequence was added to clarify this important point
Line 167 -Indicate from which strain the pili were purified -8013 SB? Cite Fig. S1c instead of S1b.

This is indicated
Line 174 -Change "the same procedure was conducted" to "binding was also tested" ("procedure" sounds like the phage selection procedure).Done Line 197-197 -Change "the effect of nanobodies on a possible neutralization of" to "the ability of nanobodies to neutralize".

Change made
Line 199 -Report nanobody concentration using the same units here and in the figure.This is now the case Line 202 -For clarity include "phagocytic", i.e. "phagocytic Kupffer cells" (and correct the spelling of Kupffer here and in Line 355).
Line 383 -Not clear what "G/ATDH" or this sentence means -reword.We meant GATDH or DATDH but GATDH is sufficient here and the text was corrected Line 404 -delete "specific" Deleted Line 474 -"during" should be "for" Modified Line 484 -"Preparation of pili" should be "Pilus preparations" or just "Pili".Changed Line 489 -"pics" should be "peaks" Corrected Line 498 -Is the alpaca's name relevant?(and Alpaca need not be capitalized).This is probably not very important but the identity of the alpaca could be relevant considering that, over the years, these animals receive successively several injections with different antigens and they have their own immunological history.
Line 517 -Correct spelling of peroxidase.Corrected Line 585 -"Camelidae" should be "Camelid".Changed Line 749/50 -Check period placements.Corrected Line 775 -Variable amino acids are shown in green -is this regardless of whether or not the changes are conserved?Recommend coloring only non-conserved changes.

Sentence has been changed to clarify
Line 823 -Correct "phospohoglycerol".Corrected Line 844 -"indicate" Corrected Fig. S4 -Some of the text is small and faint so hard to read.Text has been made larger Supplementary tables are not labeled.Something is wrong with the formatting.

The table and its format have been modified accordingly
Reviewer #3 (Remarks to the Author): This work produced the cryo-electron microscopy structures of pili of different sequence types with sufficiently high resolution to visualize posttranslational modifications and their complexes with nanobodies.Molecular dynamics simulations contributed to the structure modeling and refinement.While little simulation data was presented to validate the findings, the supplementary videos provide some details.The simulation protocol seems alright.The video S1 shows the membrane with a 16-mer pilus inserted keeps moving up, or the 16-mer keeps inserting deeper, until the final quarter of the simulation when G3P touches the membrane.This does not invalidate the simulation, but does show that the initial insertion of the pilus is not right.The pilus is actually not sinking into the lipid bilayer, rather, the pilus occasionally tilts to the side giving the impression that it is sinking depending on the angle of observation.A different angle was chosen to avoid this impression.
Fig 2 b-d: needs a befter figure legend and definifion of the scale bar.

Fig 4a :
Fig 4a: please expand the boundary of the pili, the current cropping does not allow seeing the protruding anfibodies.

Fig 5 :
Fig 5: please add a pilus and highlight which secfions are shown in a-e Fig 6a,b: please separate the two sets of structures, it is very confusing to discern the two forms Fig 6c: please change the colors or enlarge the image: the blue, red and violet are difficult to disfinguish Fig. S2a: The panel with inifial helical symmetry esfimafion has low contrast; diffracfion spots are barely seen.Please improve the contrast Fig S2c: define befter what is meant by a "middle secfion of the pilus" as it could lead to confusion Fig S5: graph is missing the x-axis label.On the results, please comment on the intermediate species around the tall peaks Fig S6 is missing Fig 2 b-d: needs a better figure legend and definition of the scale bar.Modified Fig 4a: please expand the boundary of the pili, the current cropping does not allow seeing the protruding antibodies.Modified Fig 5: please add a pilus and highlight which sections are shown in a-e A pilin monomer has been added with an indication the regions shown in a-e

Fig. S2a :
Fig. S2a: The panel with initial helical symmetry estimation has low contrast; diffraction spots are barely seen.Please improve the contrast The resolution and contrast have been improved and image made larger.